Bone is a dynamic and continuously renewed tissue that undergoes significant modification throughout the life cycle. Bone remodeling integrates osteoblast-mediated bone formation and osteoclast-mediated bone resorption as well as osteocytes within the bone matrix and osteoblast-derived lining cells that cover the surface of bone (Crockett et al., 2011; Sims and Gooi, 2008). Disrupting the balance between bone resorption and formation leads to bone disorders such as osteoporosis, multiple myeloma, and osteopetrosis (Chen et al., 2018b; Feng and McDonald, 2011; Langdahl et al., 2016; Walsh and Gravallese, 2010). Osteoporosis is defined by the World Health Organization (WHO) as a ‘progressive systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture’ (Liu et al., 2019). It is estimated that osteoporosis has affected hundreds of millions of people worldwide, predominantly postmenopausal women (Brown, 2017; Compston et al., 2019). Pharmacological agents for the treatment of osteoporosis can be classified as either anabolic or antiresorptive, in the other words, either increasing bone building or blocking bone degradation (Compston et al., 2019; Liu et al., 2016a). However, treatment with anabolic agents such as parathyroid hormone (PTH) might lead to a simultaneous increase in bone resorption. For the antiresorptive agent bisphosphonates, a concomitant decrease in bone formation can be observed (Khosla and Hofbauer, 2017; Liu et al., 2018). These drawbacks of the current therapies might be attributed to one target for these drugs that fail to couple bone formation and resorption. Researchers are constantly striving to explore dual-action agents that modulate bone remodeling for the treatment of osteoporosis (Deal, 2009).

A large number of factors have been implicated in regulating bone formation, including Wnt/β-catenin signaling pathway, which is crucial for osteoblast proliferation, differentiation, and survival (Krishnan, 2006). Activation of the canonical Wnt/β-catenin signaling pathway involves recruitment of a degrading complex, stabilization of β-catenin, regulation of transcription factors such as Runx2 and Osterix, and activation of Wnt target genes (Baron and Kneissel, 2013; Karner and Long, 2017; Takada et al., 2009). This pathway is active in osteoprogenitor cells or preosteoblasts such as MC3T3-E1 cells, and many signaling molecules in this pathway are developed as drug targets (Bhukhai et al., 2012; Zhou et al., 2020). Osteoclasts, formed by the differentiation and fusion of hematopoietic mononuclear precursor cells under the stimulation of the receptor activator of nuclear factor-κB ligand (RANKL), are the only cells responsible for bone resorption. After binding to receptor RANK, RANKL promotes osteoclastogenesis by activating several signaling pathways, including nuclear factor of activated T cells, cytoplasmic 1 (NFATc1)/c-fos pathways (Kim and Kim, 2016; Zhao et al., 2017). Furthermore, several genes such as cathepsin K, matrix metalloprotein-9 (MMP-9), and tartrate-resistant acid phosphatase (TRAP) that governing osteoclast differentiation and function are triggered after RANKL/RANK signal transduction cascades (Boyle et al., 2003; Jiang et al., 2019).

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family that regulates a variety of biological processes predominantly via binding to the transmembrane receptor tyrosine kinase B (TrkB). Nonetheless, BDNF-elicited TrkB signals are transient with a peak signal at 10 min and decreases at 60 min (Liu et al., 2016a; Liu et al., 2014). A systemic application study of BDNF found that BDNF had a short half-life and did not readily cross the blood-brain barrier (Felts et al., 2002). The outcomes of several clinical trials using recombinant BDNF are also disappointing, possibly because of the short in vivo half-life and poor delivery of BDNF (Ochs et al., 2000; Thoenen and Sendtner, 2002). These drawbacks impose an insurmountable hurdle to clinical application of BDNF. 7,8-Dihydroxyflavone (7,8-DHF), a plant-derived flavonoid, has been reported to overcome the above limitations of BDNF and identified as a functional BDNF mimic, which is able to induce TrkB dimerization and activate its downstream signaling molecules (Jang et al., 2010). 7,8-DHF is now extensively validated in various biochemical and cellular systems. It has been shown that BDNF could promote osteoblast differentiation, migration, and fracture healing (Kilian et al., 2014; Liu et al., 2018; Zhang et al., 2020; Zhang et al., 2017). Recently, we found that 7,8-DHF at the dose of 10 mg/kg·BW resulted in a marked increase in bone mineral density (BMD) irrespective of dietary conditions in female mice (Zhao et al., 2021). Inspired by the studies above, we wonder if 7,8-DHF could serve as a regulator on bone remodeling and exert antiosteoporosis effects. What’s more, it is of interest to explore whether TrkB mediates the interaction between 7,8-DHF-induced osteoblastogenesis and Wnt/β-catenin signaling. In this study, we attempted to investigate the effects of BDNF and 7,8-DHF on osteoblastogenesis in osteoprogenitor MC3T3-E1 cells and on RANKL-induced osteoclastogenesis in RAW264.7 cells in vitro, particularly to elucidate the molecular mechanisms underlying functions of 7,8-DHF. In addition, the in vivo effects of 7,8-DHF on bone remodeling were examined in an ovariectomy (OVX)-induced osteoporosis rat model.

In this study, we performed a series of studies in vitro to demonstrate that 7,8-DHF promoted osteoblastic differentiation by interacting with TrkB receptors and subsequently reinforcing the Wnt/β-catenin pathway in MC3T3-E1 cells. In addition, 7,8-DHF inhibited RANKL-induced osteoclastogenesis by suppressing c-fos in RAW264.7 cells. Moreover, the effects of 7,8-DHF in vivo were explored through an OVX-induced bone-loss rat model. Results indicated that 5 mg/kg/day 7,8-DHF could improve the bone mass, microarchitecture, and bone strength of the OVX rat by inhibiting bone resorption and enhancing bone formation in vivo. Thus, our study demonstrates that 7,8-DHF could be an attractive therapeutic agent for osteoporosis treatment (Figure 7).

Figure 7

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7,8-DHF has been identified as a promising small-molecular BDNF mimetic compound, which mimics the physiological actions of BDNF via directly binding to the extracellular domain of TrkB to trigger TrkB receptor dimerization and autophosphorylation (Jang et al., 2010). 7,8-DHF has been broadly validated in various BDNF-implicated disease models including a variety of neurological diseases, mental diseases, metabolic diseases, and obesity (Agrawal et al., 2015; Bollen et al., 2012; Chen et al., 2019; Liu et al., 2016b; Pandey et al., 2020; Zhang et al., 2014). Especially, 7,8-DHF displays robust therapeutic efficacy toward Alzheimer’s disease (AD) (Gao et al., 2016; Zhang et al., 2014). At present, there are few related studies having been done on the physiological, pharmacological, and functional activities of 7,8-DHF on osteolytic disease, for example, osteoporosis. In fact, recent studies reveal the important roles of BDNF in fracture healing. As early as in 2000, BDNF and TrkB were firstly found in bone-forming cells of mice, suggesting that they were involved in the regulation of bone formation as an autocrine and paracrine factor in vivo (Asaumi et al., 2000). In 2014, the detection of BDNF and TrkB in human fracture gap tissue during various stages of the bone formation process was reported (Kilian et al., 2014). Afterward, some researches confirmed that BDNF plays a positive role in accelerating bone union (Kauschke et al., 2018a; Kauschke et al., 2018b; Liu et al., 2018). Recently, a study provided further understanding for the underlying mechanism of BDNF in fracture healing that BDNF contributed to MC3T3-E1 migration and increased the expression level of integrin β1 (Zhang et al., 2020). Considering the fact that 7,8-DHF mimics so many aspects of BDNF, we hypothesized that there may be a regulatory network involving 7,8-DHF and bone remodeling. To our knowledge, this is the first study reporting the therapeutic potential of 7,8-DHF toward osteoporosis.

Bone homeostasis requires the coordinated recruitment of osteoblasts and osteoclasts to sites of remodeling (Phan et al., 2020). The crosstalk among bone cells is maintained and orchestrated by a complex system, which includes hormones, growth factors, and physical activity (Jiang et al., 2019). Due to the tight coupling and synchronization of bone formation and resorption, the limitation of clinically used drugs is stimulation or inhibition of both phases at the same time. For example, bisphosphonates decrease bone degradation, but also reduce bone formation. What’s worse, it may cause serious jaw and subtrochanteric fractures, which are mostly attributed to its effects that induce osteoclast cytotoxicity (Gasser, 2019; Khosla and Hofbauer, 2017; Reid, 2011). Likewise, drugs such as PTH or PTH-related protein mainly enhance the bone formation and meanwhile stimulate bone resorption (Leder, 2017). In our present study, 7,8-DHF, as well as BDNF, exhibited the ability to regulate bone remodeling by synchronously promoting osteoblastic differentiation and restraining the formation of osteoclasts. Previous studies reported that BDNF is a potential osteoclastogenic factor in multiple myeloma, and myeloma-derived BDNF could promote RANKL secretion and osteoclasts formation (Ai et al., 2012; Sun et al., 2012). Nevertheless, in our study we found that BNDF at the concentration of 25 ng/mL markedly inhibited the formation of TRAP-positive multinucleated osteoclasts derived from RAW264.7 cells, though contributed to the growth of RAW264.7 cells. Of note, we observed that the TRAP-positive cells, c-fos mRNA level, the expression of MMP-9, and Adamts5 were decreased when 0.5 or 1 μM 7,8-DHF was used, while the inhibitory effect was lost at dose of 5 μM. This observation indicated that the effective therapeutic dose range of 7,8-DHF for suppression of bone resorption might be narrow. The underlying molecular mechanism accounting for this curve remains unclear. Nevertheless, the prodrug of 7,8-DHF, R13, also displayed the similar bell-shaped pharmacological curve in therapeutic treating AD (Chen et al., 2018a). Therefore, optimization of the chemical structure of 7,8-DHF or development of its analogues might exhibit a positive dose-dependent effect, extending the inhibitory activity for osteoclastogenesis.

Besides 7,8-DHF, other natural products or their derivatives such as emodin, wedelolactone, rhein, and psoralen have also showed dual effects on bone remodeling (Jiang et al., 2019; Kim et al., 2014; Liu et al., 2016a; Newman and Cragg, 2020; Zhang et al., 2019). One of the possible causes of their dual effects might result from that they can target multiple signaling pathways and display various beneficial pharmacological effects (Jiang et al., 2019). The canonical Wnt/β-catenin signaling pathway has been demonstrated to be responsible for a variety of biological processes, including the regulation of osteoblast proliferation and differentiation. β-Catenin is the principal mediator of Wnt/β-catenin signaling pathway (Baron and Kneissel, 2013; Gordon and Nusse, 2006). In the absence of extracellular Wnt ligands, β-catenin is recruited into a degrading complex comprising GSK-3β, the scaffold protein axin, and adenomatous polyposis coli (Moon et al., 2004). Thus, the content of the β-catenin protein in normal cells is generally kept very low. In the presence of Wnt ligands, on the other hand, GSK3β is phosphorylated to yield inactive p-GSK3β, and the degrading complex is depolymerized, which allows β-catenin to avoid the degradation by the proteasome and accumulate in the cytoplasm (Chen et al., 2021; Wang et al., 2019). The accumulated β-catenin translocates into the nucleus, binds with T-cell-specific transcription factor/lymphoid enhancer binding factor, and activates a series of target gene, such as cyclin D1, Runx2, and Osterix. It has been reported that BDNF is involved in the regulation of GSK3β activity both in the retina tissue in vivo and in the neuronal cells in vitro (Gupta et al., 2014). In this research, we observed that 7,8-DHF induced phosphorylation of GSK3β at the serine nine residue, thus promoting the aggregation and localization of β-catenin in the nucleus.

Cyclin D1, whose activity is required for G1/S transition, is one of the key cell cycle regulators. It is synthesized during G1 phase and assembles with either cyclin-dependent kinase 4 (CDK4) or CDK6 (Fujita et al., 2002; Ozaki et al., 2007). Treatment of MC3T3-E1 cells with 7,8-DHF could up-regulate the mRNA level of cyclin D1, thus stimulating more cells at S phase but less at G0/G1 phase. These results indicated active DNA synthesis, which was consistent with cell proliferation results. Two transcription factors, Runx2 and Osterix/SP7, have previously been shown to be essential for the differentiation of osteoblasts (Ryoo et al., 2006; Yang and Karsenty, 2002). Runx2 not only acts as a scaffold for nucleic acids and regulatory factors involved in skeletal gene expression, but also promotes skeletal development on different levels, including differentiation of mesenchymal progenitors into osteoblasts, chondrocyte maturation, and skeletal morphogenesis (Kokabu et al., 2016; McGee-Lawrence et al., 2014). Osterix plays an important role at a later step in the process of osteoblast differentiation, that is, the differentiation of pre-osteoblasts into mature osteoblasts and osteocytes. Moreover, it is a key target of mechanical signals that affect bone biology (Fan et al., 2007). In the present study, we have shown that 7,8-DHF could enhance osteoblastic differentiation by stimulating Runx2 and Osterix via β-catenin. The possibility that 7,8-DHF enhanced the activity of other Runx2 inducers such as Smad2 and TAK1 was excluded.

What molecule(s) served as a ‘bridge’ between 7,8-DHF-induced osteogenesis and the activation of the Wnt/β-catenin pathway? It has been reported that 7,8-DHF inhibits obesity through activating muscular TrkB. The anti-obesity activity of 7,8-DHF is muscular TrkB-dependent and 7,8-DHF cannot mitigate diet-induced obesity in female muscle-specific TrkB-knockout mice (Chan et al., 2015). Besides, the promotion effect of BDNF on osteoblast migration and fracture healing is also via TrkB. The specific inhibitor for Trks, K252a, can suppress BDNF-induced cell migration, integrin β1 expression, and activation of ERK1/2 and AKT signaling pathways (Zhang et al., 2020). We thus hypothesized that TrkB is the link between 7,8-DHF and Wnt/β-catenin reinforcement during osteogenic differentiation. As expected, 7,8-DHF-induced activation of Wnt/β-catenin signaling pathway in MC3T3-E1 cells was diminished by inhibiting TrkB with K252a. In the presence of K252a, the elevation of the expression of p-GSK3β, β-catenin, Runx2, and Osterix was reduced, suggesting that 7,8-DHF acts on TrkB to alter the expression of critical genes involved in the Wnt/β-catenin signaling pathway.

Differentiated osteoblasts subsequently produce positive and negative regulators of osteoclastogenesis, such as RANKL, a member of the tumor necrosis factor-ligand family, and its natural decoy receptor OPG, respectively (Kong et al., 1999; Lacey et al., 1998). RANKL binds to its receptor RANK on osteoclast progenitors and activates c-fos to induce transcription of genes related to osteoclasts (Leibbrandt and Penninger, 2008). Whereas OPG competitively binds to RANKL and prevents activation of RANK, leading to the inhibition of osteoclast recruitment. Otherwise, the production of OPG is tightly dependent on the Wnt/β-catenin signaling pathway (Li et al., 2020). From the experimental results, we infer that 7,8-DHF-induced increase of OPG might be involved in the activation of Wnt/β-catenin signaling pathway, and the decreased RANKL probably down-regulated the transcription of c-fos to a certain degree, leading to the suppression of NF-κB pathway and ultimately inhibiting the osteoclastic differentiation of RAW264.7 cells. The RANK/RANKL/OPG axes couple osteoblast and osteoclast activities, thereby controlling the balance between bone formation and resorption (Boyle et al., 2003). The ratio between OPG and RANKL is considered a quantitative osteogenic activity standard (Liu et al., 2012). The current study suggests that 7,8-DHF positively modulates OPG/RANKL balance, as OPG/RANKL ratio was increased in response to 7,8-DHF, representing the capability of 7,8-DHF to inhibit osteoclast activity.

Considering the fascinating dual functional effect of 7,8-DHF on osteoblasts and osteoclasts in vitro, we further proved that 7,8-DHF exerted a protective effect against osteoporosis in vivo using an OVX-induced osteoporotic rat model. In agreement with the previous observations, ovarian hormone deficiency, caused by surgical removal of ovaries, significantly increased body weight (Zhao et al., 2019; Zhou et al., 2020), but this excess body weight gain was partially prevented by 7,8-DHF. Besides, OVX can accelerate bone remodeling, an uncoupling between bone resorption and formation (Libouban et al., 2003). As the resorption phase is short but the period required for osteoblastic replacement is long, any increase in the rate of bone remodeling will result in a loss of bone mass (Raisz, 2005). It is necessary to evaluate the true impact of a treatment on the quality of trabecular bone because trabecular bone is more readily lost due to OVX in this animal model (Maïmoun et al., 2012). Radiographic and histomorphology analysis showed that 7,8-DHF attenuated bone loss and alleviated the compromised trabecular microstructure in ovariectomized rat. However, neither of the doses was able to restore trabecular bone completely. The loss of bone mass is usually accompanied by enhanced levels of the bone turnover markers, such as ALP and BGP (Park et al., 2011). The relief of the high bone turnover status in the 7,8-DHF-treated groups was evidenced by lower serum levels of ALP and BGP. Moreover, an increase in urinary calcium excretions might contribute to the reduction of BMD (Park et al., 2011). Results showed that 7,8-DHF prevented the OVX-induced increase in urinary Ca excretion. Although such inhibition generally would be considered beneficial, biomechanical properties of bone may be decreased if bone remodeling is inhibited excessively. Fortunately, we found that 7,8-DHF at a lower dose (5 mg/kg/day) significantly improved the biomechanical parameters in OVX rats.

In summary, the present study evaluates the potential of 7,8-DHF as a novel dual regulator for bone formation and bone resorption. We found that 7,8-DHF promoted osteoblastic proliferation and differentiation through the TrkB-Wnt/β-catenin signaling pathway and inhibited osteoclastogenesis at the same time. Furthermore, 7,8-DHF could alleviate osteoporosis phenotypes, enhance mechanical properties, and ameliorate bone remodeling in OVX rat. Notably, 7,8-DHF is orally bioactive and is safe for chronic treatment. Hence, it acts as a good compound for further medicinal application to maximize its therapeutic properties toward various bone disorders, especially postmenopausal osteoporosis.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Fan Xue

   Department of Food Science and Nutrition, College of Biosystems Engineering and Food Science, Zhejiang Key Laboratory for Agro-Food Processing; Zhejiang Engineering Center for Food Technology and Equipment, Zhejiang University, Hangzhou, China

   Contribution

   Resources, Data curation, Formal analysis, Validation, Investigation, Visualization, Writing - original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0057-3792

2. Zhenlei Zhao

   Department of Food Science and Nutrition, College of Biosystems Engineering and Food Science, Zhejiang Key Laboratory for Agro-Food Processing; Zhejiang Engineering Center for Food Technology and Equipment, Zhejiang University, Hangzhou, China

   Contribution

   Resources, Formal analysis, Investigation, Writing - original draft

   Competing interests

   No competing interests declared

3. Yanpei Gu

   Department of Food Science and Nutrition, College of Biosystems Engineering and Food Science, Zhejiang Key Laboratory for Agro-Food Processing; Zhejiang Engineering Center for Food Technology and Equipment, Zhejiang University, Hangzhou, China

   Contribution

   Formal analysis, Investigation, Visualization

   Competing interests

   No competing interests declared

4. Jianxin Han

   Department of Food Science and Nutrition, College of Biosystems Engineering and Food Science, Zhejiang Key Laboratory for Agro-Food Processing; Zhejiang Engineering Center for Food Technology and Equipment, Zhejiang University, Hangzhou, China

   Contribution

   Formal analysis, Visualization

   Competing interests

   No competing interests declared

5. Keqiang Ye

   Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, United States

   Contribution

   Conceptualization, Resources, Supervision, Project administration, Writing - review and editing

   For correspondence

   kye@emory.edu

   Competing interests

   No competing interests declared

6. Ying Zhang

   Department of Food Science and Nutrition, College of Biosystems Engineering and Food Science, Zhejiang Key Laboratory for Agro-Food Processing; Zhejiang Engineering Center for Food Technology and Equipment, Zhejiang University, Hangzhou, China

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Project administration, Writing - review and editing

   For correspondence

   yzhang@zju.edu.cn

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8533-4138

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